The goal of regenerative medicine is to restore form and function to damaged and aging tissues. Muscle stem cells (MuSCs), also known as satellite cells, are responsible for maintenance and regeneration of skeletal muscle mass. We showed that MuSCs in aged muscles have inherent defects in their quiescence and self-renewal mechanisms, and prematurely senesce and differentiate. Our laboratory has established methods to characterize murine MuSCs by flow cytometry in conjunction with in vivo functional assays and has been able to successfully culture MuSCs ex-vivo in artificial stem cell niches that recapitulate the biophysical environment in which muscle stem cells reside. Here we propose to discern the role of extrinsic factors within or near the niche in modulating MuSC function during aging. Critical to the approach is single- cell analysis, as the behavior of slow proliferating MuSC may be masked by more rapidly proliferating progenitors and the MuSC population, though highly enriched, is heterogeneous and contains multiple subsets based on expression profiles, signaling and functional studies. We will capitalize on two single-cell technologies: (a) a bioengineered culture platform that supports maintenance of MuSC phenotype ex-vivo and long-term single-cell lineage tracking by time-lapse microscopy and (b) single cell mass cytometry, which allows high-throughput analysis of multiple biological cell surface and intracellular markers with single-cell resolution enabling an unbiased approach to the identification of novel subsets within a complex cell population. We will compare MuSCs from young and aged tissues, which have inherently different quiescence and self-renewal capacities. We aim (1) to identify extrinsic factors that regulate MuSC fate in vitro. A family of cytokines identified in a 660-peptide library of secreted and transmembrane ectodomain proteins will be assayed for their potential to alter MuSC proliferation and phenotype; (2) to characterize specific members of the family of cytokines that regulate MuSC quiescence, asymmetric, or symmetric self- renewal to alter cell fate; (3) to elucidate the MuSC subsets and signaling networks stimulated by this cytokine family. We will employ single-cell mass cytometry to resolve distinct functional subsets of MuSCs, investigate how these subsets differentially activate signaling networks in response to extrinsic quiescence, proliferation, and commitment-inducing stimulation by the cytokine family members, and determine how those differences affect MuSC contributions to regeneration and strength. Together, these studies will provide insights into the role of extrinsic factors in the stem cell microenvironment on stem cell function and suggest novel therapeutic approaches to muscle degenerative diseases and muscle aging.